Process optimization for green synthesis of silver nanoparticles using Rubus discolor leaves extract and its biological activities against multi-drug resistant bacteria and cancer cells

Multi-drug resistant (MDR) bacteria are considered a serious public health threat. Also, increasing rate of resistance to anticancer drugs, as well as their toxicity, is another point of concern. Therefore, the new antibacterial and anticancer agents are always needed. The synthesizing silver nanoparticles (AgNPs) using medicinal plants, is an effective approach for developing novel antibacterial and anticancer agents. Rubus discolor, a native species of the Caucasus region, produces leaves that are typically discarded as a by-product of raspberry production. The present study has focused on optimizing the green synthesis of AgNPs using R. discolor leaves extract through response surface methodology. The optimal values for AgNPs synthesis were an AgNO3 concentration of 7.11 mM, a time of 17.83 h, a temperature of 56.51 °C, and an extract percentage of 29.22. The production of AgNPs was confirmed using UV–visible spectroscopy (λmax at 456.01 nm). TEM analysis revealed well-dispersed AgNPs (an average size of 37 nm). The XRD analysis confirmed the crystalline structure. The EDX detected a strong peak at 3 keV corresponded to Ag. The zeta potential value (− 44.2 mV) indicated the stability of nanoparticles. FT-IR spectra showed the presence of various functional groups from plant compounds, which play an important role in the capping and bio-reduction processes. The AgNPs revealed impressive antibacterial activities against MDR Escherichia coli and Pseudomonas aeruginosa (MIC ranging from 0.93 to 3.75 mg ml−1). The phytochemical analysis indicated the presence of phenolics, tannins, and flavonoids on the surface of AgNPs. They also showed significant cytotoxic effects on A431, MCF-7, and HepG2 cells (IC50 values ranging from 11 to 49.1 µg ml−l).

industry 8 .According to their shapes, sizes, and properties, NPs have been classified into several groups, including carbon-based nanoparticles, polymeric nanoparticles, ceramic nanoparticles, and metal nanoparticles 9 .Among them, metal nanoparticles comprise gold, silver, copper, magnetic (cobalt, iron, and nickel), and semiconducting materials 2,10 .Silver nanoparticles (AgNPs) have gained significant attention based on various biological activities, including, antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and wound healing properties 11,12 .They have been used in food processing, cosmetics, home cleaning, catalytic and garment production, and pharmaceutical industry 13 .
There are various mechanical and chemical methods for producing nanomaterial that have several disadvantages, such as high cost, low yield, and being relatively complicated.Also, they are not environmentally friendly due to the application of toxic chemicals and solvents, and the production of dangerous by-products 6,9,14 .However, the green approaches for NPs production are eco-friendly, easy to apply, and safe for the environment, human beings, and living organisms 11,15 .Therefore, there is an increasing demand for developing safe methods to produce nanomaterials, using fungi, bacteria, or plants 6,[16][17][18][19] .The plant extracts are preferred over other natural materials.Many natural compounds have been discovered in medicinal plants that play a crucial role in the preparation of nanoparticles 12 .The plant's secondary metabolites, such as phenolic compounds, tannins, flavonoids, anthraquinones, carbohydrates, alkaloids, alkynes, allylic benzenes, ascorbic acids, alcoholic compounds, sugars, amides, amino acid residues, carotenes, steroids, saponins, and triterpenoids have proven to be able to reduce silver nitrate to AgNPs 7,12,20 .Recent investigations have revealed that NPs synthesized by bioactive phytochemicals possess more beneficial and effective properties than traditional herbal drugs 12 .
The World Health Organization (WHO) has recently declared antibacterial resistance as one of the three main risks to human health 21 .Drug-resistant pathogens kill about 700,000 people worldwide each year, and this number could increase to 10 million deaths a year by 2050 22 .Presently, the rapid development of drug-resistant strains of microorganisms as well as the serious lack of effective antibiotics, have revealed that the discovery and development of novel antimicrobial agents are logically necessary 22,23 .Silver is an inorganic antibacterial agent, which is nontoxic and safe.It is effective against 650 types of pathogenic microorganisms 24 .Studies showed that AgNPs can exert significant antibacterial activity against MDR (multi-drug resistant) bacteria by several mechanisms, such as inhibition of the cell respiration chain, disrupting the cellular signal transduction pathways, and generating reactive oxygen species (ROS), which causes toxicity in cells 12,[25][26][27] .
Moreover, cancer is the second leading cause of death universally after cardiovascular diseases 28 .The American Cancer Society predicted that the worldwide burden of cancer will surge to 21.7 million fresh cases by the year 2030 29 .Today, there is growing attention to discovering inexpensive and more cost-effective drugs using natural resources like medicinal plants.The plants have been provided various new approaches for the cancer treatment, including green synthesis of AgNPs 29 .
Blackberries, Rubus spp., Rosaceae family, are widely distributed and cultivated worldwide and are of growing commercial relevance 30 .This genus comprises over 750 species in the world.The sweet taste fruits of many species are popular as a healthy and nutritious food, containing various phenolic compounds, dietary fiber, vitamin C, α-tocopherol, and carotenoids 31,32 .Also, blackberry leaves have been used traditionally, in form of tea or as a mouthwash, and gargle solution.It is reported that blackberry leaves have important bioactive components, such as phenols, flavonoids, tannins, terpenoids, and other anti-aging and antioxidant compounds, and can serve as a potential source for use in the food, pharmaceutical industry, and cosmetic 32,33 .In the flora of Iran, eight species of blackberry have been reported 34 .Rubus discolor Weihe & Nees., commonly known as Himalayan blackberry, is a native species of the Caucasus region of Eurasia 35 .It is widely distributed in the North and Northwest of Iran as a common weed 36 .Since the Rubus leaves are significantly consumed less than fruits, a large number of leaves are disposed of as a by-product of raspberry production 32 .Currently, the assessment of bioactive phytochemicals in the eliminated plant material has attracted great interest, because these by-products have high levels of constituents with biological properties 37 .As a result, the ecofriendly synthesis of AgNPs from the leaves which have potential biological activities could be of interest.
In the present study, the aqueous extract of Rubus discolor leaves was used for the biosynthesis of the AgNPs.The synthesis condition was optimized by response surface methodology (RSM).The nanoparticles were characterized by UV-Vis and FT-IR spectroscopic methods, as well as XRD, DLS, SEM-EDX, and TEM methods.The preliminary phytochemical investigations and the determination of total phenolic, tannin, and flavonoid contents were performed.The antibacterial activity was tested against two ATCC Gram-positive (Streptococcus aureus and Bacillus subtilis) and two ATCC Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa).Also, ten MDR isolates of E. coli and P. aeruginosa were tested to investigate their susceptibility to the synthesized AgNPs.The cytotoxic activities of AgNPs and extract were investigated against three cancerous cell lines, including MCF-7 (breast cancer), A431 (epidermoid carcinoma), and HepG2 (liver hepatocellular carcinoma) as well as a normal cell line (HU02) by MTT assay.

Phytochemical investigation of the aqueous extract and AgNPs
The preliminary phytochemical analysis of the aqueous extract of leaves revealed the presence of flavonoids, tannins, steroids, and carbohydrates (Table 1).
The total phenolic content (TPC) of the aqueous extract and AgNPs were calculated based on the gallic acid standard curve equation (y = 0.000899x − 0.0355, R 2 = 0.999), using the Folin-Ciocalteu method.Also, the total flavonoid contents (TFC) were measured, based on the quercetin standard curve (y = 0.0192x − 0.0198, R 2 = 0.995).The total tannin contents (TTC) were measured using the following standard curve plotted for tannic acid (as standard compound): y = 0.005x + 0.0281; R 2 = 0.995.All the results are depicted in Table 2.According to the results, AgNPs showed lower TPC, TTC, and TFC than the aqueous leave extract.The reduction in polyphenolic

Statistical process optimization of green synthesis AgNPs using RSM
The results of the central composite design (CCD) for optimizing Ag synthesis conditions, including AgNO 3 concentration, time, temperature, and the extract percent, were represented in Table 3.
A set of 30 runs based on the formula 2 N + 2 N + X was conducted, where N is the number of selected factors with 2 N factorial (16 runs), 2N axial (8), and X center points repetitions (6 runs).The Eq. 1 shows the correlation between the absorbance at 456 nm (as an indicator of SPR) and the four studied parameters in coded terms: where Y is the absorbance at 456 nm; A is AgNO 3 concentration; B is the time of reaction; C is temperature; and D is the extract percentage.The analysis of variance (ANOVA) was carried out to investigate the suitability of the obtained model (Table 4).
Based on the statistics, a quadratic model was suggested to relate the experimental factors and their combinations and the response.The high F-value (30.34) and the low p value (p < 0.0001, only 0.01% chance of noise) showed that the obtained model is significant and acceptable.The variables A, B, C, D, AD, A 2 , B 2 , C 2 , and D 2 were the significant parameters on the basis of p value (p < 0.05).The F-value lack of fit was 3.38 with a p value of 0.1723 (chance of noise 17.23%), which shows the model is valid.
Three parameters, including the calculated determination coefficient (R 2 and adjusted R 2 ) and adequate precision, were used to evaluate the model's efficacy of R 2 and adjusted R 2 values of 0.9703 and 0.9383, respectively, showed that the model has high efficacy and can properly explain the variability.Adequate precision (AP) of 19.305 (AP > 4 is desirable), which shows the signal-to -noise ratio, indicated adequate signal-to-noise.The Predicted R 2 of 0.7957 showed a high correlation between predicted and observed responses.It should be in reasonable agreement with adjusted R 2 (within the range of 0.2 adjusted R 2 ).
For evaluation of the best condition for each factor to obtain the maximum AgNPs yield, the 3D surface and contour plots were used (Fig. 1).These plots were on the basis of the corresponding interactions of two factors, while the third parameter was fixed at the optimum condition.The shape of the 3D contour plot shows the interaction significance.
Figure .1a-c shows that AgNO 3 concentration (A) had a significant effect on the AgNPs synthesis.When AgNO 3 concentration increased, the yield of AgNPs increased depending on the second parameter.Othman et al. reported that AgNO 3 concentration strongly affected the yield of AgNPs synthesis when interacting with other factors such as reaction pH value 40 .Likewise, El-Rafie showed that increasing the silver nitrate concentration dramatically increased the absorbance intensity 41 .Figures 1a, e, and f show that the time of the reaction (B) had a lesser momentous influence on yield of AgNPs synthesis in the interaction with AgNO 3 concentration (A), temperature (C), and extract percent (D).When the reaction time increased, the AgNPs biosynthesis increased by the interaction of the second factor, including A, C, or D. Figures 1b, d, and f prove that temperature (C) had a stronger effect on the AgNPs biosynthesis than the time of reaction.Also, in a study, the AgNPs were synthesized using Plantago major extract, and it was showed that temperature had higher effect on the absorbance in comparison with time 42 .Figures 1c, e, and f explain that extract percent (D) had the second rank in the AgNPs (1) Y = 1.77 + 0.34A + 0.11B + 0.2C + 0.23D + 0.036AB − 0.014AC + 0.19AD + 0.03BC www.nature.com/scientificreports/synthesis after AgNO 3 concentration.The yield of AgNPs biosynthesis increased with the increase in extract percent due to higher reducing agents in the reaction mixture 43 .A strong interaction was observed between A and D, and other interactions were not significant.The 3D surface plots showed that the effects of the four studied parameters on the AgNPs green synthesis were not equal, and the order of factors was as follows: A > D > C > B, respectively.In the optimized condition, the selected experimental model was tested using AgNO 3 concentration of 7.11 mM, time of 17.83 h, temperature of 56.51, and extract percent of 29.22.The predicted absorbance at 456 nm was 1.92, which is close to the experimental value (2.12) that indicates the validity of the models.The yield of the reaction, in optimized condition was 53.31%.

Characterization of AgNPs Optical properties and UV-Vis spectroscopy of the synthesized AgNPs
The formation of AgNPs was first characterized by the observation of color change from pale yellow to dark brown, that revealed the Ag + reduction into Ag 0 nanoparticles.The color transformation is due to AgNPs' optical properties and known as the localized surface plasmon resonance (SPR) 44 .Various factors like particle type, size, shape, morphology, dielectric environment, and composition have an impact on SPR.Also, UV-Vis spectroscopy is a common characterization tool to detect the SPR absorption peak of NPs and demonstrate their formation 45 .As depicted in Fig. 2, the UV-V is spectrum of AgNPs showed a SPR at 456.01 nm.In a study, Said et al. reported that the UV-vis spectrum of the AgNPs they produced was observed at 460 nm 16 .Additionally, Patra et al. revealed that their AgNPs had a maximum absorbance peak at 456 nm 46 .These findings are consistent with our study.The numerous phytochemicals present in the aqueous extract of R. discolor could be responsible for the rapid bioreduction and capping of synthesized AgNPs.Typically, the bioactive compounds such as vitamins, flavonoids, tannins, phenolic acids, proteins, etc., are responsible for the fast reduction of Ag + , and control the size distribution and morphology of synthesized NPs 47 .According to the results of this study, the leaves extract contained tannins, flavonoids, steroids, and carbohydrates, which can act as reducing agents.Table 3. Coded values of used independent variables in response surface central composite design matrix and the observed response (absorbance) for AgNPs.www.nature.com/scientificreports/

TEM analysis
The AgNPs were evaluated by the transmission electron microscopes (TEM) for elucidation of the size, shape, and morphology.The microphotographs displayed that the nanoparticles were well-distributed and roughly spherical, with polydispersity, and without agglomeration.The size of the most particles ranged between 20 and 50 nm, with an average size of 37 nm (Fig. 3).It can be suggested that during the reaction, the content of reducing agent in plant extract deceased gradually, which led to the formation of AgNPs in different sizes.Also, careful observation of TEM images revealed no direct connection among AgNPs, even within the aggregates,  www.nature.com/scientificreports/presenting that AgNPs were surrounded by a thin layer of natural phytochemicals like amino acids and phenolic compounds 7,38 .In a study, Mariadoss et al. reported that the morphology of the AgNPs synthesized by the extract of Malus domestica was spherical, with polydispersity and a size ranging from 40 to 100 nm 48 .Also, Yassin et al. synthesized AgNPs from Origanum majorana, which showed polydispersity, with a size range from 10 to 60 nm 8 .

SEM-EDX analysis
The surface nature and the elemental configuration of the AgNPs were determined by Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray (EDX) analysis.The SEM image displayed that AgNPs were  www.nature.com/scientificreports/polydisperse, about 38 nm in size, and predominantly spherical in shape (Fig. 4).The EDS analysis of the AgNPs was conducted to study the elemental composition of AgNPs.EDX analysis displayed a strong signal at 3.0 keV, which is characteristic of metallic Ag because of surface plasmon resonance, associated with the Ag-L a line 49 .Additionally, the profile exhibited peaks for oxygen and carbon which could be attributed to the phytoconstituents attached to the surface of the AgNPs.Our results were in accordance with those of Okaiyeto et al., which produced AgNPs using aqueous extract of Oedera genistifolia, showing the presence of intense peak of silver element at 3.0 keV 50 .Moreover, Patra et al. synthesized AgNPs from Pisum sativum.The EDX spectra of their study showed the elemental composition of the AgNPs, with a strong peak at 3 keV that corresponded to Ag 46 .

Zeta potential and DLS analysis
The particle size distribution of biosynthesized AgNPs was determined using a dynamic light scattering (DLS).
The DLS determines the hydrodynamic size of colloids, and can estimate the average size of the nanoparticles in the mixture, approximately 51 .The results of this study indicated that the particle size and poly-dispersity index (PDI) values of AgNPs were 151.7 nm and 0.25, respectively (Fig. 5).As reported in previous studies, PDI (also known as heterogeneity index) shows the non-uniformity of particles in a colloidal solution.This value is unitless and is considered between 0.05 and 0.7.A PDI value close to 0.05 indicates that the particles are monodispersed, while colloidal solutions with PDI values close to 0.7 are heterogenous 51,52 .In this study, the reported PDI value of 0.25 is acceptable.On the other hand, the zeta potential (ζ) is one of the important factors for the characterization of the stability of nanoparticles in a solution.Nanoparticles with zeta potentials larger than + 30 mV and less than − 30 mV show considerable stability for colloidal dispersions 51 .The value of AgNPs zeta potential was − 44.2 mV.The highly negative value of ζ proved that the synthesized AgNPs had high stability 53 .Since, in this study, the external stabilizers were not used, meaning that the plant phytochemicals acted not only as the reducing agents of the Ag + to Ag 0 , but also stabilized the synthesized nanoparticles.
It is obvious that the particle size in the DLS analysis is larger than in the TEM test.The DLS measurement is carried out in a fluid phase.This means that the AgNPs particles are in constant movement because of Brownian motion.Also, AgNPs have a charge on their surfaces, and consequently, they can interact with other ions, molecules, and surfaces, which contributes to the creation of adsorbed layers on the surface of the nanoparticles 14 .Therefore, the DLS shows the hydrodynamic diameter of the biomolecules surrounding AgNPs and the intensityweighted average particle size 44 .However, the TEM image is taken in a dry state.Thus, the results of DLS and TEM cannot be in line with each other, and the results of DLS showed a normally larger size than those of TEM 14 .

X-Ray Diffraction Spectroscopy
The X-ray diffraction (XRD) pattern of AgNPs synthesized using aqueous extract of R. discolor is shown in Fig. 6.The peaks at 2 theta (θ) degrees of 38.1°, 44.2°, 64.5°, and 77.6° could be related to (111), ( 200), (220), and (311) facets, respectively, which corresponded to the database of the Joint Committee on Powder Diffraction Standards (JCPDS), file No. 00-004-0783.Debye-Scherrer formula (Eq.2) was used to calculate the size of AgNPs, as follow: where D is the average crystallin size of AgNPs, λ is the wavelength of X-ray which is 0.1546 nm, β is the width at half maximum of the peak in radians, and θ is Braggs angle in degrees 54 .Similar to previous studies, it shows

FT-IR analysis
FT-IR analysis was performed to determine the structure of phytochemicals, exciting in aqueous leaves extract of R. discolor, which are responsible for surface coating and stabilization of the AgNPs.Figure 7 shows the IR spectra of the aqueous extract and the synthesized AgNPs.The wavenumbers of different functional groups are summarized in Table 5.The characteristic peaks are determined by comparing peaks with the FT-IR results of other studies that have biosynthesized AgNPs using green methods.
By comparison the FT-IR spectra of AgNPs and the leaves aqueous extract, it was demonstrated that some peaks were shifted.Also, the intensity of some peaks reduced or increased, and the appearance of several new peaks changed significantly.For example, peaks at 3228, 1601, 1516, and 1380 cm −1 , corresponding to O-H and N-H stretching vibrations, shifted to 3160, 1596, 1513, and 3151 cm −1 , respectively.That could be due to some electrostatic interactions among the AgNPs and functional groups of capping agents.Moreover, in the FT-IR spectrum of AgNPs, the appearance of a peak at 1632 cm -1 , as well as increasing the intensity of the peak at 1436 cm -1 , which are attributed to carbonyl vibrations, designated that the reduction of the silver ions is due to the oxidation of the hydroxyl groups to the carbonyl groups in the plant extract.The reduced peak intensity at 3160 cm −1 revealed the important role of OH and N-H in the reduction and binding mechanism 7 .Finally, new peaks at 596 and 494 cm −1 may be attributed to the bonding of AgNPs with phytochemicals in the extract.Similar to our study, Said et al. reported peaks at 775 to 540 cm −1 which were correspondent to the bonding of AgNPs to functional groups in the extract 16 .Also, Aref et al. suggested that the peaks at 488 and 407 cm −1 may refer to the binding of AgNPs to phytochemical groups 55 .bdel-Raouf et al. 56 Yousefbeyk et al. 7 Salem S.  www.nature.com/scientificreports/

Suggested mechanism of formation of AgNPs
The plant extract contains various molecules such as polyphenols, terpene derivatives, saccharides, alkaloids, etc.These molecules are responsible for the reduction of AgNO 3 to Ag 0 .The probable mechanism of AgNPs synthesis is depicted in Fig. 8. Generally, the functional groups such as hydroxyl (-OH) of these biomolecules interact with AgNO 3 .When AgNO 3 dissolves in water, it dissociates into two ions, Ag + and NO 3 − .The acidic nature of OH groups of phytochemicals resulted in donation of H + ions and acquisition of a negative charge.The negative functional groups like O − of phenols interact electrostatically with Ag + .This process leads to the reduction of Ag + ions.The NO 3 − ions accept H + from phenolic OH resulted in the formation of HNO 3 .Ag remains in a free metallic state (Ag 0 ) to form AgNPs 50 .

Antibacterial activity
Green synthesized AgNPs (at concentration of 1 mg ml −1 ) displayed significant antibacterial activity against ATCC gram-negative bacteria, including P. aeruginosa and E. coli, with inhibition zone of 18 and 16.5 mm, respectively.However, the AgNPs did not show any antibacterial activity against gram-positive pathogens (Fig. 9).The aqueous extract of R. discolor exhibited no antibacterial effect even in a high concentration (300 mg ml −1 ).Also, the best MIC value was for P. aeruginosa ATCC (0.83 mg ml −1 ) (Table 6).
Also, the antibacterial activity was measured against MDR E. coli and P. aeruginosa isolated.Results of the antibiogram susceptibility test of eight antibiotics against ten isolates are depicted in Table 7.
Also, Fig. 10 describes the zone of inhibition (mm) for eleven antibiotics and AgNPs against isolate number 5 of MRD E. coli and P. aeruginosa.As is presented in Table 8, the AgNPs showed antibacterial activity against MDR E. coli with MIC values ranged from 1.87 to 3.75 mg ml −1 .The MBCs were 5 mg ml −1 for all of the MDR E. coli isolates.Moreover, the MIC values against MDR P. aeruginosa isolated ranged from 0.93 to 1.87 mg ml −1 , and the MBC values were 2.5-5 mg ml −1 .The aqueous extract did not have any antibacterial activity against tested MDR isolates.
AgNPs had ultra-small size and uniform distribution that led to significant antibacterial activity 7 .It is proposed that AgNPs release Ag + that attach to the negative charge of the microbial cell wall, denaturing the membrane proteins.Also, AgNPs have potent affinity for the sulfur-containing proteins in the cell wall, leading to changes in the morphological structure of the cell membrane.This irreversible damage increases the permeability of the cell membrane, thereby disrupting the cell ability to regulate normal activity.This can lead to the loss or leakage of cellular contents such as, proteins, cytoplasm, ions, and cellular energy sources 57 .After crossing the cell membrane, AgNPs disturb the bacteria's metabolic pathways.They cause several intracellular changes like enzyme inhibition, interaction with bacterial DNA resulting in denaturation of DNA, interruption of the bacteria growth, and inducing electrolyte imbalance 7,15,47 .Another mechanism of action is increasing oxidative stress by inducing overproduction of ROS (reactive oxygen species).ROS can oxidate macromolecules like lipids, DNA, and proteins and therefore, cause the bacterial death (Fig. 11).
Studies have been shown that AgNPs are more effective against gram-negative bacteria strains than grampositive ones.The suggested reason is that the gram-positive bacteria consist of one cytoplasmic membrane and a relatively thick cell wall that include numerous peptidoglycan layers (thickness between 20 and 80 nm).In contrast, gram-negative strains, there is an external layer of lipopolysaccharide (LPS) as well as one thin layer of peptidoglycan and an internal plasma membrane 58 .Our results are in consistence with previous reports.

Cytotoxic assay
The anti-proliferative effects of silver NPs and the leaves aqueous extract were investigated against three human cancerous cell lines and a healthy cell line.The IC 50 of AgNPs on selected cancerous cell lines ranged from 11.2 to 49.1 µg ml −1 (Fig. 12).The silver NPs exhibited more cytotoxic activities on MCF-7 and A431 cells than on HepG2 cells.Also, AgNPs showed more potent anti-proliferative activity than the aqueous extract on all cancerous cell line, particularly on HepG2 that the cytotoxicity of AgNPs was 2.5 times more than crude extract.Furthermore, the cytotoxic effect of AgNPs was investigated on HU02 (a noncancerous cell line).It was revealed that AgNPs had much less cytotoxic activity against the normal cell line (IC 50 of 158 µg ml −1 ) in comparison with the extract.www.nature.com/scientificreports/Recently, AgNPs have attracted great attention for their possible use as an anticancer therapeutic agent because of their significant cytotoxic effect on cancerous cell lines, while they are less toxic on normal cell lines 7,38 .It is suggested that the Ag + , released from AgNPs, can directly bind to RNA polymerase, disturbing its activity.Another main proposed mechanism of cytotoxicity is the generation of ROS, which leads to intracellular oxidative stress and consequently cell death.It has been observed that the cytotoxicity of AgNPs is size-dependent.The smaller AgNPs can more easily penetrate the cell membrane and interact with different cell parts.Also, it has been reported that the AgNPs, with higher surface area, can sustainably release more concentration of silver cations 38,59 .It has been revealed that the green synthesized AgNPs can carry numerous plant secondary metabolites on their surface that enhance the effectiveness of AgNPs 7,38 .Table 9 summarizes the IC 50 values of AgNPs prepared from several plant extracts against the same cancerous cell lines as our study.As is presented, the IC 50 values of AgNPs synthesizes from leaves of R. discolor was in the range of previous studies.Also, AgNPs from Table 6.Antimicrobial activities of the AgNPs and R. discolor leaves extract against ATCC bacteria strain.ZI zone of inhibition (mm); MIC minimum inhibitory concentration (mg ml −1 ); MBC minimum bactericidal concentration (mg ml −1 ); a -: no antibacterial activity reported; the results are the mean ± SD.

AgNPs extract AgNPs extract AgNPs extract
Staphylococcus aureus (ATCC 6538)   R. discolor had stronger cytotoxic activities against MCF-7, and A431 compared to the AgNPs that are prepared from other mentioned plant extracts in Table 9.

Conclusion
In current study, the biosynthesized AgNPs were characterized using UV-Vis spectroscopy, FT-IR analysis, DLS, TEM, SEM-EDX, and XRD.All these characterizations confirmed the synthesis of AgNPs with average size of 37 nm.The results of the FTIR spectra showed that the phytochemicals present in R. discolor extract play a key role in the production of AgNPs.Phytochemical analysis showed that the leaves of R. discolor are a good source of phytochemicals, including phenolics, tannins, and flavonoids.Besides, having health-beneficial effects, these compounds have the ability to reduce silver ions, along with surface coating and stabilization of the AgNPs.The study also aimed to optimize the physical parameters and discover the interaction relations between variables affecting AgNPs biosynthesis, using RSM.The experimental results exhibited that all the factors studied were significant for the variable responses.The optimized condition was found to be an AgNO 3 concentration of 7.11 mM, a time of 17.83 h, a temperature of 56.51 °C, and an extract percentage of 29.22, with a yield of 53.31%.Considering the surge in antibiotic resistance, the AgNPs prepared from R. discolor can be potentially used as an antibacterial agent against MDR E. coli and P. aeruginosa pathogens (MIC 0.93-3.75mg ml −1 ).The AgNPs depicted significant cytotoxicity against A431, MCF7, and HepG2 (IC 50 11.2-49.1 µg ml −1 ), while no significant  www.nature.com/scientificreports/toxicity against normal cell line was observed.This optimized, low-cost, and environmentally-friendly method is a valuable approach for producing bioactive silver NPs with high yield and small size.

Plant materials and extraction
The aerial parts of R. discolor were collected from Fuman-Saravan Road, Guilan province, in the North of Iran, in May 2021 (Fig. 13).The voucher specimen (113 HGUM) was kept in the herbarium of the faculty of pharmacy, Guilan University of Medical Sciences, Rasht, Iran.The plant's leaves were separated from the stems.Then, leaves were shade-dried at room temperature for two weeks and powdered with a mixer grinder.Consequently, 100 g of powder were added to 600 ml of deionized water (DW) and boiled for 15 min.After that, the mixture was cooled and filtered through the Whatman filter paper 7 .Lastly, the solvent was evaporated using a rotary vacuum evaporator (Heidolph, Germany) at 45 °C to obtain 4.1 g of dried extract.It was kept in the refrigerator at 4ºC until required.

Preliminary phytochemical tests
The preliminary qualitative phytochemical assays were carried out to identify the presence of secondary metabolites in the extract, including flavonoids, tannins, anthraquinones, steroids, carbohydrates, coumarins, and alkaloids, using the standard protocols described by Saeidnia & Ghohari 67 .

Determination of total phenolic content (TPC)
The Folin-Ciocalteu method was used to measure the total phenolic contents in the extract and AgNPs 68 .In this test, 1 ml of each sample (1 mg ml −1 ) was added to 5 ml of freshly prepared Folin-Ciocalteu reagent (diluted tenfold with distilled water).Then, the mixtures were incubated for 10 min at room temperature before mixing with 4 ml sodium bicarbonate solution (75 g l −1 ).They were incubated for 30 min in the dark.Lastly, the absorbance was obtained at 765 nm using a UV/Vis spectrophotometer.All the experiments were repeated three times.The gallic acid (GA) was used as the reference standard in different concentrations (10, 25, 50, 100, and 150 µg ml −1 ), and the calibration curve was plotted.The total phenolic contents were expressed as mg of gallic acid equivalents (GAE)/g extract.

Determination of total flavonoid content (TFC)
The measurement of the flavonoid was carried out by the Dowd method 67,68 .First, aluminum trichloride (AlCl 3 ) (2%) was prepared in methanol.Then, 5 ml of AlCl 3 solution was added to 5 ml of each sample (2 mg ml −1 ).The mixtures were incubated for 10 min at room temperature.Finally, the absorbance was measured at 415 nm using a UV/Vis spectrophotometer 7 .All the experiments were repeated three times.The quercetin was used as the www.nature.com/scientificreports/standard compound with five known concentrations (10, 25, 50, 75, and 100 µg ml −1 ).Finally, the total flavonoid content was expressed as mg of quercetin as equivalents (QE)/g of extract.

Determination of total tannin content (TTC)
The aqueous extract and synthesized AgNPs were examined for the total tannin contents by a colorimetric method using polyvinylpolypyrrolidone (PVPP) [69][70][71] .In this assay, PVPP binds to tannins and precipitates them.Different concentrations of tannic acid (20, 40, 60, 80, 100, 150, and 200 μg ml −1 ) were used for plotting the calibration curve.This method involved two steps.In the first step, 1 ml of each sample (1 mg ml −1 ) was combined with 0.5 ml Folin-Ciocalteu reagent (1 N).Next, sodium carbonate solution (2.5 ml, 20%) was added to each mixture.After 40 min, the absorbance was read at 725 nm.The amounts of total phenols as tannic acid equivalent (X) were calculated using the calibration curve.In the second step, the tannins were removed from tannincontaining samples by adding PVPP (100 mg of PVPP is adequate to bind 2 mg of total phenols).The samples were vigorously shaken (5 min) and kept at 4 °C (15 min).After that, the samples were centrifuged at 4000 g (20 min), and the supernatants were collected.The supernatant only contained simple phenols other than tannins.
The phenolic contents of the supernatants were measured, as explained in the first step.The contents of nontannin phenols (Y) were determined.Lastly, X-Y showed mg of tannin as tannic acid equivalent (TAE)/g extracts.

Green synthesis of AgNPs
In a typical reaction procedure, different amount of aqueous extract was added to five different concentrations of AgNO 3 solution (50 ml, 1-10 mM), based on CCD described in the next section.The mixtures were stirred on a magnetic stirrer (Heidolph, Germany) at a constant rate (500 rpm) at different times and temperatures.Next, the mixtures were centrifuged for 15 min at 10,000 rpm using a centrifuge machine 7 .Finally, the sediments were washed three times with deionized water, and dried in a vacuum oven (45 °C).The yield of the AgNPs formation was calculated in optimized condition.

Experimental design and optimization of AgNPs synthesis by RSM
Previous studies showed that different parameters like concentration of extract and AgNO 3 , time, and temperature have great influence on the size and yield of synthesized AgNPs 72 .In this study, a central composite design (CCD) under Response Surface Methodology (RSM) was employed for the optimization of the most prominent parameters and also for the identification of their cooperative interactions using Design-Expert 7.0 (Stat-Ease, Inc., USA software).Four independent variables were selected, including reaction time (h), reaction temperature (°C), AgNO 3 concentration, and percentage of extract (%).Each variable was evaluated at five coded levels (− 2, − 1, 0, 1, 2) (Table 10).The total experimental runs were calculated using the following equation: 2 k + 2k + x 0 , where k is a variable number and x 0 is the repetition number of experiments at the center point 40 .

Figure 2 .
Figure 2. UV-Vis spectrum of the R. discolor extract and AgNPs.

Figure 3 .
Figure 3. TEM images of AgNPs at optimized condition.

Figure 5 .
Figure 5. Size (a) and zeta potential value (b) of AgNPs prepared by R. discolor.

Figure 8 .
Figure 8. Suggested reduction mechanism of Ag + to Ag 0 .

Figure 11 .
Figure 11.The antibacterial mechanism of action of AgNPs.

Table 4 .
ANOVA analysis of CCD for the synthesized AgNPs.

Table 5 .
FT-IR analysis of R. discolor extract and the green synthesized AgNPs displaying different functional groups.

Table 8 .
The Zone of inhibitions, MIC, and MBC values of AgNPs on E. coli and P. aeruginosa isolates.E E. coli; P P. aeruginosa; Zone of inhibition (mm); MIC minimum inhibitory concentration (mg ml −1 ); MBC minimum bactericidal concentration (mg ml −1 ).

Table 9 .
The comparison of cytotoxic activities of green synthesized AgNPs from leaves of R. discolor with other synthesized AgNPs on MCF-7, HepG2, and A431.

Table 10 .
Selected levels of experimental variable in building the CCD.